Analyte detection

ABSTRACT

This disclosure relates generally to detection and, in particular, to detecting objects within a sample or fraction thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/946,498, filed Apr. 5, 2018.

FIELD

This disclosure relates generally to detection and, in particular, todetecting objects within a sample or fraction thereof.

BACKGROUND

Samples often include objects that are to be imaged for analysis. Theseobjects may include a plurality of biomarkers and/or components forwhich it is desirous to detect and image. As a result, practitioners,researchers, and those working with samples continue to seek systems andmethods to more efficiently and accurately detect and image objects of asample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an example method including two antigen retrieval steps.

FIG. 2 shows example images for example objects.

DETAILED DESCRIPTION

In the following descriptions, the term “light” is used to describevarious uses and aspects of multiplexing and imaging. The term light isnot intended to be limited to describing electromagnetic radiation inthe visible portion of the electromagnetic spectrum, but is alsointended to describe radiation in the ultraviolet and infrared portionsof the electromagnetic spectrum.

In the following descriptions, the term “sample” is used to describe abiological fluid, a biological semi-solid, a biological solid (which mayremain solid, such as tissue, or may be liquefied in any appropriatemanner), a suspension, a portion of the suspension, a component of thesuspension, or the like. For example, for anticoagulated whole blood,the sample is the anticoagulated whole blood (i.e. a suspension), thebuffy coat (i.e. a portion of the suspension), or a circulating tumorcell (i.e. a component of the suspension). For the sake of convenience,the sample referenced is whole blood, though it should be understoodthat the method and system described and discussed herein is used withany appropriate sample, such as urine, blood, bone marrow, buffy coat,cystic fluid, ascites fluid, stool, semen, cerebrospinal fluid, nippleaspirate fluid, saliva, amniotic fluid, mucus membrane secretions,aqueous humor, vitreous humor, vomit, vaginal fluid, and any otherphysiological fluid or semi-solid. For example, the sample is a tissuesample or a material from adipose tissue, an adrenal gland, bone marrow,a breast, a caudate, a cerebellum, a cerebral cortex, a cervix, auterus, a colon, an endometrium, an esophagus, a fallopian tube, a heartmuscle, a hippocampus, a hypothalamus, a kidney, a liver, a lung, alymph node, an ovary, a pancreas, a pituitary gland, a prostate, asalivary gland, a skeletal muscle, skin, a small intestine, a largeintestine, a spleen, a stomach, a testicle, a thyroid gland, or abladder.

In the following descriptions, the terms “target analyte” or “targetmaterial” are used to describe a biological material of interest. Itshould also be understood that the target analyte can be a fraction of asample, such as buffy coat, a cell, such as ova, fetal material (such astrophoblasts, nucleated red blood cells, fetal red blood cells, fetalwhite blood cells, fetal DNA, fetal RNA, or the like), a circulatingtumor cell (“CTC”), a circulating endothelial cell, an immune cell (i.e.naïve or memory B cells or naïve or memory T cells), a mesenchymal cell,a stem cell, a vesicle, such as an exosome, a liposome, a protein, anucleic acid, a biological molecule, a naturally occurring orartificially prepared microscopic unit having an enclosed membrane,parasites (e.g. spirochetes, such as Borrelia burgdorferi which causeLyme disease; malaria-inducing agents), microorganisms, viruses, orinflammatory cells. For example, the target analyte is a tumor cell fromadipose tissue, an adrenal gland, bone marrow, a breast, a caudate, acerebellum, a cerebral cortex, a cervix, a uterus, a colon, anendometrium, an esophagus, a fallopian tube, a heart muscle, ahippocampus, a hypothalamus, a kidney, a liver, a lung, a lymph node, anovary, a pancreas, a pituitary gland, a prostate, a salivary gland, askeletal muscle, skin, a small intestine, a large intestine, a spleen, astomach, a testicle, a thyroid gland, or a bladder.

In the following descriptions, the term “non-target analyte” is used todescribe a biological material which is not a target analyte.

In the following descriptions, the term “biomarker” is used to describea substance that is present on or within the target analyte or targetmaterial (i.e. intracellular or extracellular the target analyte;internalized, such as through phagocytosis, within the target analyte;or the like). Biomarkers include, but are not limited to, peptides,proteins, subunits, domains, motifs, epitopes, isoforms, DNA, RNA, orthe like. The biomarker may be a target molecule for drug delivery.

In the following descriptions, the term “affinity molecule” is used todescribe any molecule that is capable of binding or interacting with abiomarker. The interaction or binding can be covalent or non-covalent.The affinity molecule includes, but is not limited to, an antibody, ahapten, a protein, an aptamer, an oligonucleotide, a polynucleotide, orany appropriate molecule for interacting with or binding to thebiomarker.

In the following descriptions, the term “detection moiety” is used todescribe a compound or substance which provides a signal for detection,thereby indicating the presence of another compound or substance, ananalyte, or the like within a sample or specimen. The detection moietycan be fluorescent, such as a fluorescent probe, or chromogenic, such asa chromogenic dye.

In the following descriptions, the term “channel” is used to describe acolor or color range based on the signal provided by one or moredetection moieties. The color or color range is obtained based on thefilters chosen and/or the wavelength of the signal(s). For example, achannel may be violet, blue, green, yellow, orange, red, dark red, orthe like. Furthermore, when a plurality of channels are used, eachchannel has a specific color or color range. For example, a firstchannel may be green and a second channel may be orange. It should benoted that although two or more detection moieties may provide signalshaving different wavelengths, the signals can be in the same channelbased on the filter set used. For example, a first detection moietyprovides signal having a wavelength of 488 and a second detection moietyprovides a signal having a wavelength of 500. Even though thewavelengths are not identical, the filter set in one of the channelspasses wavelengths of both 488 nm and 500 nm, which permits both to beimaged at the same time, thereby producing a single image including the488 and 500 emissions.

In the following descriptions, the terms “stain” or “label,” which areused interchangeably, are used to describe an affinity molecule bound toor interacted with a detection moiety. The binding or interaction can bedirect or indirect. Direct binding or interaction includes covalent ornon-covalent interactions between the biomarker and the detectionmoiety. Indirect binding or interaction includes the use of at leastfirst and second complementary molecules which form binding pairs. Thefirst and second complementary molecules are, in combination, bindingpairs which binds or interacts in at least one of the following manners:hydrophobic interactions, ionic interactions, hydrogen bondinginteractions, non-covalent interactions, covalent interactions, affinityinteractions, or the like. The binding pairs include, but are notlimited to, immune-type binding-pairs, such as, antigen-antibody,antigen-antibody fragment, hapten-anti-hapten, or primaryantibody-secondary antibody; nonimmune-type binding-pairs, such asbiotin-avidin, biotin-streptavidin, folic acid-folate binding protein,hormone-hormone receptor, lectin-specific carbohydrate, enzyme-enzyme,enzyme-substrate, enzyme-substrate analog, enzyme-pseudo-substrate(substrate analogs that cannot be catalyzed by the enzymatic activity),enzyme-cofactor, enzyme-modulator, enzyme-inhibitor, or vitaminB12-intrinsic factor. Other suitable examples of binding pairs includecomplementary nucleic acid fragments (including complementarynucleotides, oligonucleotides, or polynucleotides); Protein A-antibody;Protein G-antibody; nucleic acid-nucleic acid binding protein; polymericlinkers (e.g., polyethylene glycol); or polynucleotide-polynucleotidebinding protein. The binding pairs can be included within or used asamplification techniques. Amplification techniques are also implementedto increase the number of detection moieties bound to or interacted withthe biomarker to increase a signal. In one embodiment, when bindingpairs are used, the stain can be pre-conjugated, such that, during alabeling, staining, or adding step, the affinity molecule is alreadybound to or interacted with a detection moiety when added to the sample.In one embodiment, when binding pairs are used, the stain can beconjugated in the sample, such that the labeling, staining, or addingstep includes at least two sub-steps including introducing (in anydesired or appropriate order) an affinity molecule-first bindingmolecule conjugate and a second binding pair molecule-detection moietyconjugate, wherein the first and second binding pair molecules arecomplementary and bind to or interact with each other.

Furthermore, “a plurality of stains” can be used to describe two or morestains in which the affinity molecules and/or the detection moieties aredifferent. For example, anti-CK-Alexa 647 is different thananti-EpCAM-Alexa 647. As another example, anti-CK-Alexa 647 is differentthan anti-CK-Alexa 488.

In the following descriptions, the terms “permeabilize” or“permeabilization” are used to describe the dissolution or removal of aportion of a plasma membrane of a target or non-target analyte bychemical or other means, such that at least an IgG antibody is capableof crossing the plasma membrane.

In the following descriptions, the term “conjugate” is used to describea first chemical, molecule, moiety, or the like bound to or interactedwith a second chemical, molecule, moiety, or the like. The binding orinteraction is direct or indirect. Direct binding or interactionincludes covalent or non-covalent interactions between the biomarker andthe detection moiety. Indirect binding or interaction includes the useof at least first and second complementary molecules which form bindingpairs. The first and second complementary molecules are, in combination,binding pairs which binds or interacts in at least one of the followingmanners: hydrophobic interactions, ionic interactions, hydrogen bondinginteractions, non-covalent interactions, covalent interactions, affinityinteractions, or the like. The binding pairs include, but are notlimited to, immune-type binding-pairs, such as, antigen-antibody,antigen-antibody fragment, hapten-anti-hapten, or primaryantibody-secondary antibody; nonimmune-type binding-pairs, such asbiotin-avidin, biotin-streptavidin, folic acid-folate binding protein,hormone-hormone receptor, lectin-specific carbohydrate, enzyme-enzyme,enzyme-substrate, enzyme-substrate analog, enzyme-pseudo-substrate(substrate analogs that cannot be catalyzed by the enzymatic activity),enzyme-cofactor, enzyme-modulator, enzyme-inhibitor, or vitaminB12-intrinsic factor. Other suitable examples of binding pairs includecomplementary nucleic acid fragments (including complementarynucleotides, oligonucleotides, or polynucleotides); Protein A-antibody;Protein G-antibody; nucleic acid-nucleic acid binding protein; polymericlinkers (e.g., polyethylene glycol); or polynucleotide-polynucleotidebinding protein.

General Method

An example method for labeling a biomarker on a target analyte isdiscussed. In one embodiment, a sample, such as blood, is obtained, suchas by venipuncture. The sample is suspected of including at least onetarget analyte. Suitable devices, systems, and/or methods of samplecollection and/or processing may include those described in one or moreof the following U.S. patents and published applications, each of whichis hereby incorporated by reference in its entirety: U.S. Pat. Nos.7,074,577; 7,220,593; 7,329,534; 7,358,095; 7,629,176; 7,915,029;7,919,049; 8,012,742; 9,039,999; 9,217,697; 9,492,819; 9,513,291;9,533,303; 9,539,570; 9,541,481; 9,625,360; 2014/0161688; 2017/0014819;2017/0059552; 2017/0074759. Suitable devices, systems, and/or methodsfor target analyte retrieval, isolation, or picking may include thosedescribed in one or more of the following U.S. patents and publishedapplications, each of which is hereby incorporated by reference in itsentirety: U.S. Pat. Nos. 9,222,953; 9,440,234; 9,519,002; 9,810,605;2017/0219463; 2017/0276575.

The sample is then dispensed onto or into at least one analysisplatform. The analysis platform is a microscope slide, a positivelycharged microscope slide, a negatively charged microscope slide, acoated microscope slide, a porous slide, a micro-well slide, a wellplate, a coverslip, a cell microarray, or the like. The analysisplatform can be any appropriate material, including, but not limited to,glass, plastic, ceramic, metal, or the like.

In one embodiment, the sample is re-suspended in an attachment solutionin a vessel prior to being dispensed onto the analysis platform. Forexample, the attachment solution is added to or mixed with the sample.The re-suspended sample, which includes at least a portion of theattachment solution is dispensed onto or into the analysis platform by adispenser, such as a pipet or repeating pipet. In one embodiment, theattachment solution includes an alcohol (such as ethanol or methanol)and a non-steroidal anti-inflammatory drug (such as aspirin). In oneembodiment, the attachment solution includes an alcohol (such as ethanolor methanol) and an anti-coagulant (such as heparin). In one embodiment,the attachment solution includes an alcohol (such as ethanol ormethanol), an anti-coagulant (such as heparin), and a non-steroidalanti-inflammatory drug (such as aspirin).

In one embodiment, the sample is spread across the analysis platform. Inone embodiment, the sample is spread across the analysis platform by aspreader, such as a squeegee, a pipet tip, a blade, a two-piece spreaderincluding a blade and a base. In one embodiment, the sample is spreadacross the analysis platform by centrifuging, wetting, or nutating theanalysis platform.

In one embodiment, the re-suspended sample is cured to adhere there-suspended sample to the analysis platform. In one embodiment, there-suspended sample is dispensed onto the analysis platform and curedwithout being spread across the analysis platform. Curing occurs in air,such as at room temperature; in an environmentally-controlled chamber,such as at 37° C.; or the like. Furthermore, the sample may undergo anadditional fixation step, such as in treatment with formalin or anyappropriate fixative, after the curing step has been completed.

In one embodiment, at any point prior to staining, autofluorescence isreduced or eliminated. The autofluorescence is reduced or eliminated bybleaching the sample with a chemical (such as hydrogen peroxide), anenzyme, light, heat, or the like.

The sample then undergoes staining. At least one stain is added to thesample for labeling, such as by an autostainer or manually by anoperator. In one embodiment, the at least one target analyte is stained.In one embodiment, at least one non-target analyte or non-targetmaterial is stained. In one embodiment, the at least one target analyteand the at least one non-target analyte or materials are stained.

The biomarker or biomarkers include, but are not limited to: 17-IA,4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 Adenosine Receptor,A33, ACE, ACE-2, Activin, Activin A, Activin AB, Activin B, Activin C,Activin RIA, Activin RIA ALK-2, Activin RIB ALK-4, Activin RIIA, ActivinRIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAM8, ADAM9, ADAMTS,ADAMTS4, ADAMTS5, Addressins, aFGF, ALCAM, ALK, ALK-1, ALK-7,alpha-1-antitrypsin, alpha-V/beta-1 antagonist, ANG, Ang, APAF-1, APE,APJ, APP, APRIL, AR, ARC, ART, Artemin, anti-Id, ASPARTIC, Atrialnatriuretic factor, av/b3 integrin, Axl, b2M, B7-1, B7-2, B7-H,B-lymphocyte Stimulator (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1,BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM,BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 Osteogenin, BMP-4 BMP-2b,BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA(ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs, b-NGF,BOK, Bombesin, Bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC,complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8,Calcitonin, cAMP, carcinoembryonic antigen (CEA), carcinoma-associatedantigen, Cathepsin A, Cathepsin B, Cathepsin C/DPPI, Cathepsin D,Cathepsin E, Cathepsin H, Cathepsin L, Cathepsin 0, Cathepsin S,Cathepsin V, Cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12,CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21,CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6,CCL7, CCL8, CCL9/10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5,CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8,CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20,CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD31, CD32, CD33(p67 proteins), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52,CD54, CD55, CD56, CD61, CD64, CD66b, CD66e, CD74, CD80 (B7-1), CD89,CD95, CD105, CD123, CD137, CD138, CD140a, CD144, CD146, CD147, CD148,CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Clostridium botulinum toxin,Clostridium peffringens toxin, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1,COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL,CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10,CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2,CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin, DAN, DCC, DcR3, DC-SIGN, Decayaccelerating factor, des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DLL3,DNAM-1, Dnase, Dpp, DPPIV/CD26, Dtk, EGAD, EDA, EDA-A1, EDA-A2, EDAR,EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, ER,Enkephalinase, eNOS, Eot, eotaxin1, EpCAM, Ephrin B2/EphB4, EPO, ERCC,E-selectin, ET-1, Factor IIa, Factor VII, Factor VIIIc, Factor IX,fibroblast activation protein (FAP), fibronectin, Fas, FcR1, FEN-1,Ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, Fibrin, FL,FLIP, Flt-3, Flt-4, Follicle stimulating hormone, Fractalkine, FZD1,FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6,GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14,CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8(Myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1,GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, Glucagon, Glut 4, glycoproteinIIb/IIIa (GP IIb/IIIa), GM-CSF, gp130, gp72, GRO, Growth hormonereleasing factor, Hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gBenvelope glycoprotein, HCMV) gH envelope glycoprotein, HCMV UL,Hemopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2/neu(ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gBglycoprotein, HSV gD glycoprotein, HGFA, High molecular weightmelanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp120 V3loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin,human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309,IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF,IGF binding proteins, IGF-1R, IGFBP, IGF-1, IGF-II, IL, IL-1, IL-1R,IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10,IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha,INF-beta, INF-gamma, Inhibin, iNOS, Insulin A-chain, Insulin B-chain,Insulin-like growth factor 1, integrin alpha2, integrin alpha3, integrinalpha4, integrin alpha4/beta1, integrin alpha4/beta7, integrin alpha5(alphaV), integrin alpha5/beta1, integrin alpha5/beta3, integrin alpha6,integrin beta1, integrin beta2, interferon gamma, IP-10, I-TAC, JE,Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein 11, Kallikrein 12,Kallikrein 14, Kallikrein 15, Kallikrein L1, Kallikrein L2, KallikreinL3, Kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), Ki67,laminin 5, LAMP, LAP, LAP (TGF-1), Latent TGF-1, Latent TGF-1 bp1, LBP,LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1,LFA-3, Lfo, LIF, LIGHT, lipoproteins, LIX, LKN, Lptn, L-Selectin, LT-a,LT-b, LTB4, LTBP-1, Lung surfactant, Luteinizing hormone, LymphotoxinBeta Receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP,M-CSF, MDC, Mer, METALLOPROTEASES, MGDF receptor, MGMT, MHC (HLA-DR),MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11,MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8,MMP-9, MPIF, Mpo, M30, MSK, MSP, mucin (Muc1), MUC18,Muellerian-inhibitin substance, Mug, MuSK, NAIP, NAP, NCAD, N-Cadherin,NCA 90, NCAM, NCAM, Neprilysin, Neurotrophin-3,-4, or -6, Neurturin,Neuronal growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3,NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr,Parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-Cadherin, PCNA,PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2,placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, Proinsulin,Prorelaxin, Protein C, PR, PS, PSA, PSCA, prostate specific membraneantigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES,Relaxin A-chain, Relaxin B-chain, renin, respiratory syncytial virus(RSV) F, RSV Fgp, Ret, Rheumatoid factors, RLIP76, RPA2, RSK, 5100,SCF/KL, SDF-1, SERINE, Serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM,SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI,TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T-cell receptors(e.g., T-cell receptor alpha/beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8,TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha,TGF-beta, TGF-beta Pan Specific, TGF-beta RI (ALK-5), TGF-beta R11,TGF-beta RIIb, TGF-beta RIII, TGF-beta1, TGF-beta2, TGF-beta3,TGF-beta4, TGF-beta5, Thrombin, Thymus Ck-1, Thyroid stimulatinghormone, Tie, TIMP, TIQ, Tissue Factor, TMEFF2, Tmpo, TMPRSS2, TNF,TNF-alpha, TNF-alpha beta, TNF-beta2, TNFa, TNF-RI, TNF-RII, TNFRSF10A(TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2DR5, KILLER, TRICK-2A,TRICK-B), TNFRSF10C (TRAIL R3DcR1, LIT, TRID), TNFRSF10D (TRAIL R4DcR2,TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF11B (OPG OCIF, TR1),TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14(HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17(BCMA), TNFRSF18 (GITRAITR), TNFRSF19 (TROY TAJ, TRADE), TNFRSF19L(RELT), TNFRSF1A (TNF R1CD120a, p55-60), TNFRSF1B (TNF RII CD120b,p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1,CD95), TNFRSF6B (DcR3M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9(4-1 BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DCTRAIL R2TNFRH2),TNFRST23 (DCTRAIL R1 TNFRH1), TNFRSF25 (DR3Apo-3, LARD, TR-3, TRAMP,WSL-1), TNFSF10 (TRAIL Apo-2 Ligand, TL2), TNFSF11 (TRANCE/RANK LigandODF, OPG Ligand), TNFSF12 (TWEAK Apo-3 Ligand, DR3Ligand), TNFSF13(APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14(LIGHT HVEM Ligand, LTg), TNFSF15 (TL1A/VEGI), TNFSF18 (GITR Ligand AITRLigand, TL6), TNFSF1A (TNF-a Conectin, DIF, TNFSF2), TNFSF1 B (TNF-bLTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (0X40 Ligand gp34, TXGP1),TNFSF5 (CD40 Ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas LigandApo-1 Ligand, APT1 Ligand), TNFSF7 (CD27 Ligand CD70), TNFSF8 (CD30Ligand CD153), TNFSF9 (4-1BB Ligand CD137 Ligand), TP-1, t-PA, Tpo,TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferring receptor, TRF,Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA 125,tumor-associated antigen expressing Lewis Y related carbohydrate, TWEAK,TXB2, Ung, uPAR, uPAR-1, Urokinase, VCAM, VCAM-1, VECAD, VE-Cadherin,VE-cadherin-2, VEFGR-1 (fit-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM,Vimentin, Viral antigens, VLA, VLA-1, VLA-4, VNR integrin, vonWillebrands factor, WIF-1, WNT1, WNT2, WNT2B/13, WNT3, WNT3A, WNT4,WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B,WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD,and receptors for hormones and growth factors. The biomarkers includeproteins, subunits, domains, motifs, isoforms, and/or epitopes belongingto the list above.

The detection moiety is a compound or substance which provides a signalfor detection, thereby indicating the presence of another compound orsubstance, an analyte, or the like within a sample or specimen. Thedetection moiety can be used as a tracer, as a label for certainstructures, as a label for biomarkers, or the like. The detection moietycan be distributed or can label the appropriate structure or biomarkersin manners including, but not limited to, uptake, selective uptake,diffusion, and attachment to a linking molecule. The detection moiety isbound to the biomarker via an affinity molecule (i.e. an antibodycapable of binding or interacting with the biomarker; or, “directlabeling”) or to the biomarker via an affinity molecule and at least onelinking molecules extending from the affinity molecule to the detectionmoiety (i.e. “indirect labeling”).

The detection moiety can be fluorescent, such as a fluorescent probe, orchromogenic, such as a chromogenic dye. The chromogenic dye, which canbe used with various enzyme labels (e.g. horseradish peroxidase andalkaline phosphate), includes, but is not limited to,3,3′-Diaminobenzidine (DAB), 3-Amino-9-Ethylcarbazole (AEC),4-Chloro-1-Naphtol (CN), P-Phenylenediamine Dihydrochloride/pyrocatechol(Hanker-Yates reagent), Fast Red TR, New Fuchsin, Fast Blue BB, or thelike. The fluorescent probe can be a reactive dye, an organic dye, afluorescent protein, a quantum dot, non-protein organic molecules, ananoparticle (e.g., nanodiamond), or the like. Fluorescent probesinclude, but are not limited to 1,5 IAEDANS; 1,8-ANS;4-Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein;5-Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein;5-Carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5-Carboxyfluorescein);5-HAT (Hydroxy Tryptamine); 5-Hydroxy Tryptamine (HAT); 5-ROX(carboxy-X-rhodamine); 5-TAMRA (5-Carboxytetramethylrhodamine);6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-Amino-4-methylcoumarin;7-Aminoactinomycin D (7-AAD); 7-Hydroxy-4-methylcoumarin;9-Amino-6-chloro-2-methoxyacridine; AB Q; Acid Fuchsin; ACMA(9-Amino-6-chloro-2-methoxyacridine); Acridine Orange; Acridine Red;Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin(Photoprotein); AutoFluorescent Protein; Alexa Fluor 350™; Alexa Fluor430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™; Alexa Fluor568™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor660™; Alexa Fluor 680™; Alizarin Complexon; Alizarin Red;Allophycocyanin (APC); AMC; AMCA-S; AMCA (Aminomethylcoumarin); AMCA-X;Aminoactinomycin D; Aminocoumarin; Aminomethylcoumarin (AMCA); AnilinBlue; Anthrocyl stearate; APC (Allophycocyanin); APC-Cy7; APTRA-BTC;APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B;Astrazon Yellow 7 GLL; Atabrine; ATTO-TAG™ CBQCA; ATTO-TAG™ FQ;Auramine; Aurophosphine G; Aurophosphine; BAO9(Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); BerberineSulphate; Beta Lactamase; BFP blue shifted GFP (Y66H; Blue FluorescentProtein); BFP/GFP FRET; Bimane; Bisbenzamide; Bisbenzimide (Hoechst);bis-BTC; Blancophor FFG; Blancophor SV; BOBO™-1; BOBO™-3; Bodipy492/515; Bodipy 493/503; Bodipy 500/510; Bodipy 505/515; Bodipy 530/550;Bodipy 542/563; Bodipy 558/568; Bodipy 564/570; Bodipy 576/589; Bodipy581/591; Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy Fl;Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; BodipyTMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-XSE; BO-PRO™-1; BO-PRO™-3; Brilliant Sulphoflavin FF; Brilliant Violet421; Brilliant Violet 510; Brilliant Violet 605; Brilliant Violet 650;Brilliant Violet 711; Brilliant Violet 786; BTC; BTC-5N; Calcein;Calcein Blue; Calcium Crimson™; Calcium Green; Calcium Green-1; CalciumGreen-2; Calcium Green-5N; Calcium Green-C18; Calcium Orange; CalcofluorWhite; Carboxy-X-hodamine (5-ROX); Cascade Blue™; Cascade Yellow;Catecholamine; CCF2 (GeneBlazer); CFDA; CFP (Cyan Fluorescent Protein);CF405S; CF488A; CF 488; CF 543; CF 647; CF 750; CF 760; CF 780; FP/YFPFRET; Chlorophyll; Chromomycin A; Chromomycin A; CL-NERF; CMFDA;Coelenterazine; Coelenterazine cp; Coelenterazine f; Coelenterazine fcp;Coelenterazine h; Coelenterazine hcp; Coelenterazine ip; Coelenterazinen; Coelenterazine 0; Coumarin Phalloidin; C-phycocyanine; CPMMethylcoumarin; CTC; CTC Formazan; Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8;Cy5.5™; Cy5™; Cy7™; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); CyQuantCell Proliferation Assay; Dabcyl; Dansyl; Dansyl Amine; DansylCadaverine; Dansyl Chloride; Dansyl DHPE; DAPI; Dapoxyl; Dapoxyl 2;Dapoxyl 3; DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR(Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS; DiA (4-Di-16-ASP);Dichlorodihydrofluorescein Diacetate (DCFH); DiD-Lipophilic Tracer; DiD(DiIC18(5)); DIDS; Dihydorhodamine 123 (DHR); DiI (DiIC18(3));Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DiIC18(7)); DM-NERF (high pH);DNP; Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP (Enhanced BlueFluorescent Protein); ECFP (Enhanced Cyan Fluorescent Protein);EGFP(Enhanced Green Fluorescent Protein); ELF 97; Eosin; ER-Tracker™Green; ER-Tracker™ Red; ER-Tracker™ Blue-White DPX; Erythrosin;Erythrosin ITC; Ethidium Bromide; Ethidium homodimer-1 (EthD-1);Euchrysin; EukoLight; Europium (III) chloride; EYFP (Enhanced YellowFluorescent Protein); Fast Blue; FDA; FIF (Formaldehyde InducedFluorescence); FITC; FITC Antibody; Flazo Orange; Fluo-3; Fluo-4;Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold(Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43™; FM 4-46; Fura Red™(high pH); Fura Red™/Fluo-3; Fura-2, high calcium; Fura-2, low calcium;Fura-2/BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF;Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer (CCF2); GFP (S65T);GFP red shifted (rsGFP); GFP wild type, non-UV excitation (wtGFP); GFPwild type, UV excitation (wtGFP); GFPuv; Gloxalic Acid; Granular Blue;Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS;Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine;Indo-1, high calcium; Indo-1, low calcium; Indodicarbocyanine (DiD);Indotricarbocyanine (DiR); Intrawhite Cf JC-1; JO-JO-1; JO-PRO-1;LaserPro; Laurodan; LDS 751; Leucophor PAF; Leucophor SF; Leucophor WS;Lissamine Rhodamine; Lissamine Rhodamine B; Calcein/Ethidium homodimer;LOLO-1; LO-PRO-1; Lucifer Yellow; Lyso Tracker Blue; Lyso TrackerBlue-White; Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker Yellow;LysoSensor Blue; LysoSensor Green; LysoSensor Yellow/Blue; Mag Green;Magdala Red (Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5;Mag-Indo-1; Magnesium Green; Magnesium Orange; Malachite Green; MarinaBlue; Maxilon Brilliant Flavin 10 GFF; Maxilon Brilliant Flavin 8 GFF;Merocyanin; Methoxycoumarin; Mitotracker Green; Mitotracker Orange;Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane(mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene);mStrawberry; NBD; NBD Amine; Nile Red; Nitrobenzoxadidole;Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant lavinEBG; Oregon Green™; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7;PerCP; PerCP-Cy5.5; PE-TexasRed (Red 613); Phloxin B (Magdala Red);Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R;PhotoResist; Phycoerythrin B; Phycoerythrin R; PKH26 (Sigma); PKH67;PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3;Primuline; Procion Yellow; Propidium lodid (PI); Pyrene; Pyronine;Pyronine B; Pyrozal Brilliant Flavin 7GF; QD500; QD525; QD625; QD800;QSY 7; Quinacrine Mustard; Red 613 (PE-TexasRed); Resorufin; RFP; RH414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD;Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; RhodamineBB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; RhodaminePhalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phycocyanine;R-phycoerythrin; rsGFP (red shifted GFP (S65T)); S65A; S65C; S65L; S65T;Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron BrilliantRed 4G; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; sgGFP™(super glow GFP; SITS (Primuline); SITS (Stilbene IsothiosulphonicAcid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF1; SodiumGreen; SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ(6-methoxy-N-(3-sulfopropyl)quinolinium); Stilbene; Sulphorhodamine Bcan C; Sulphorhodamine G Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange;SYTOX Red; Tetracycline; Tetramethylrhodamine (TRITC); Texas Red™; TexasRed-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; ThiazoleOrange; Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; ThiozoleOrange; Tinopol CBS (Calcofluor White); TMR; TO-PRO-1; TO-PRO-3;TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5);TetramethylRodaminelsoThioCyanate; True Blue; TruRed; Tubulin Tracker™Green; Ultralite; Uranine B; Uvitex SFC; wt GFP (wild type GFP); WW 781;X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP (Yellowshifted); Green Fluorescent Protein; YFP (Yellow Fluorescent Protein);YO-PRO-1; YO-PRO-3; YOYO-1; YOYO-3; and, combinations and derivativesthereof. In one embodiment, the detection moiety, such as organicfluorophore, can have a molecule weight of at least 1 kD, including,without limitation, at least 10 kD, at least 25 kD, at least 50 kD, atleast 75 kD, at least 100 kD, at least 150 kD, at least 200 kD, at least250 kD, at least 300 kD, at least 340 kD, at least 350 kD, at least 500kD, and at least 750 kD.

It should be noted that a nuclear stain, such as Sytox, may be used incombination with a quantum dot. In one embodiment, the quantum dot hasan emission peak less than or equal to 520 nm. In one embodiment, thequantum dot has an emission peak greater than or equal to 520 nm.

After labeling, the sample is imaged, whereby the sample is illuminatedwith one or more wavelengths of excitation light from a light source,such as infrared, red, blue, green, and/or ultraviolet. The imaging canbe done with a flow cytometer or a microscope, such as a fluorescentmicroscope, a scanner, or the like. Imaging can be done in brightfieldand/or darkfield illumination, phase contrast, differential interferencecontrast, fluorescence, light sheet microscopy, super resolutionmicroscopy, confocal microscopy, and Hoffman modulation contrast. Theimages formed can be overlaid when a plurality of detection moieties areused. Emission, reflection, diffraction, scatter, and combinationsthereof are used in for detection/imaging. The images are analyzed todetect, enumerate, and locate the target analyte. Imaging is performedin a tube, on a microscope slide, or in any appropriate vessel orsubstrate for imaging.

The target analyte can be retrieved from the rest of the sample. Toretrieve the target analyte, the target analyte undergoes enrichmentand/or isolation. The target analyte is isolated from rest of thesample, whether with or without prior enrichment, by selecting thetarget analyte at a time with any appropriate device or system. Imagingthe analysis platform, as discussed above, is performed to aid inisolation by providing location and characterization information forisolation purposes.

The target analyte can undergo post-processing analysis, such assequencing, by using any appropriate method or technique, though morespecifically extracellular and intracellular analysis includingintracellular protein labeling; nucleic acid analysis, including, butnot limited to, DNA arrays, expression arrays, protein arrays, and DNAhybridization arrays; or in situ hybridization (“ISH”—a tool foranalyzing DNA and/or RNA, such as gene copy number changes); polymerasechain reaction (“PCR”); reverse transcription PCR. Sequencing is done onthe entire genome, the transcriptome, or cDNA.

The steps of the method described above can be performed by at least oneof an imaging microscope, a scanner, a flow cytometer, or a microfluidicdevice, such as a chip or a microchannel, or the method can be performedby any combination of the above. For example, the flow cytometer is usedfor the collecting step, where enrichment via fluorescent-activated cellsorting is appropriate, and the microfluidic device is used for theretrieving step. In other words, the methods, though described toinclude one device per method, may be performed such that a combinationof devices are used. The devices are capable of being used in anyappropriate transmitted light modality. The devices are capable of beingused in at least one of brightfield and/or darkfield illumination, phasecontrast, differential interference contrast, fluorescence, and Hoffmanmodulation contrast imaging or detection.

In one embodiment, the patient sample is a fraction of a suspension,such that the sample is obtained through enrichment, including positiveand/or negative enrichment and/or density-based enrichment. The enrichedfraction is suspected of including at least one target analyte. Thesample is enriched by any appropriate enrichment process including, butnot limited to, sequential density fractionation, magnetic-activatedcell sorting, fluorescence-activated cell sorting, differential lysis,depletion filters, microfluidic device separation, or the like.Sequential density fractionation is a process by which a patient sampleis divided into fractions or a fraction of a sample is divided intosub-fractions by a step-wise or sequential process, such that each stepor sequence results in the collection or separation of a differentfraction or sub-fraction from the preceding and successive steps orsequences. In other words, sequential density fractionation providesindividual sub-populations of a population or individualsub-sub-populations of a sub-population of a population through a seriesof steps. For example, separation fluids are used whereby eachseparation fluid has a different density, thereby separating a fractionof a sample into sub-fractions based on the densities of the respectivesub-fractions via the different density separating fluids.

Multiplexing

Though “a biomarker” is discussed, any number of biomarkers can belabeled for multiplexing purposes, which allows, for example, multiplebiomarkers to be identified using a single image. For example, it can bedesirous to detect more than one biomarker of a given target analyte. Aset of stains, which includes at least two stains and can include up to4, 6, 8, 10, or more, is used to multiplex the target analyte, such thateach stain labels a different biomarker of the target analyte. Thisallows for detection of a plurality of biomarkers on the target materialat any given time. In one embodiment, a plurality of detection moietiesis used. For example, Alexa 488 and Alex 647 are used. In oneembodiment, a plurality of types of detection moieties are used. Forexample, Alexa 488 and QD800 are used. In one embodiment, a plurality ofdetection moieties and a plurality of types of detection moieties areused. In one embodiment, at least two stains are in the same channel andat least one stain is in at least one other channel.

Furthermore, the types of affinity molecules used are selected based onthe desired characteristics of the target analyte or in trying todetermine a particular characteristic of the target analyte. Forexample, mesenchymal, epithelial, and confirmatory/characterization(i.e. cancer cell type (e.g., prostate, breast, colon, lung, etc.))affinity molecules can be selected to characterize a given targetanalyte into various cell types. In one embodiment, a plurality ofaffinity molecules (i.e. first and second affinity molecules) isselected to bind to or interact with a plurality of biomarkers of thetarget analyte, where each of the plurality of affinity molecules is:(1) directed to a different biomarker of the target analyte; and (2)bound to detection moieties which provide signals in different channels.The detection moieties may be the same or different types. Accordingly,any number of affinity molecules and corresponding detection moietiesare implemented whereby the complementary molecules or complexes arespecific to each other. For example, an EpCAM antibody is selected asthe first affinity molecule to bind or interact with an EpCAM biomarkerof the target analyte, and a HER2 antibody is selected as the secondaffinity molecule to bind or interact with a HER2 biomarker of thetarget analyte. The EpCAM antibody is bound to or interacted with afirst detection moiety and the HER2 antibody is bound to or interactedwith a second detection moiety, such that the first and second detectionmoieties emit in a different channel.

In one embodiment, a plurality of affinity molecules (i.e. first andsecond affinity molecules) is selected to bind to or interact with aplurality of biomarkers on the target analyte, where each of theplurality of affinity molecules is: (1) directed to a differentbiomarker of the target analyte; and (2) bound to detection moietieswhich provide a signal in the same channel. The detection moieties ofthe first and second affinity molecules may be the same or may bedifferent but provide signals within the same channel. For example, anEpCAM antibody is selected as the first affinity molecule to bind orinteract with an EpCAM biomarker of the target analyte, and acytokeratin antibody is selected as the second affinity molecule to bindor interact with a cytokeratin biomarker of the target analyte. TheEpCAM antibody and the cytokeratin antibody are bound to detectionmoieties which provide a signal in, for example, the green channel.

In one embodiment, (1) a plurality of affinity molecules (i.e. first andsecond affinity molecules) is selected to bind to or interact with aplurality of biomarkers on the target analyte, where each of theplurality of affinity molecules is: (a) directed to a differentbiomarker of the target analyte, and (b) bound to the same type ofdetection moiety; and (2) at least one affinity molecule (i.e. a thirdaffinity molecule) is selected to bind to or interact with a thirdbiomarker and is bound to a different type of detection moiety. Forexample, the detection moieties bound to or interacted with the firstand second affinity molecules provide signals within the same channel,whereas the detection moiety bound to or interacted with the thirdaffinity molecules provides a signal in a different channel.

In one embodiment, a plurality of stains can be used such that eachstain includes a different binding pair. In one embodiment, a pluralityof stains can be used such that at least stains include the same bindingpair.

Cyclic Labeling

In one embodiment, multiple rounds of labeling is performed for cycliclabeling purposes. For example, a target analyte is labeled with a firstset of labels, the first set of labels are imaged, the signal providedby the stains are reduced or eliminated (such as with heat (between50-110° C., including 70° C., 80° C., 90° C., 95° C., 100° C., or 105°C.), light, chemicals, enzymes, higher affinity molecules to remove thedetection moiety, or any appropriate manner in which to reduce oreliminate the signal, such as by degrading, cleaving, or modifying), thetarget analyte is labeled with a second set of labels, and so on until adesired number of biomarkers have been labeled and/or a desired numberof rounds have been performed. The heating step is performed in anydevice or system appropriate for heating the sample, including, but notlimited to, an autostainer, a steamer (such as a vegetable steamer or abottle sanitizer), a pressure cooker, an autoclave, a water bath, a hotplate, a crockpot, a hair dryer, an incubator, a microwave, or acombination thereof.

In one embodiment, a single stain is used for each round of labeling,such that five stains are used individually across five rounds oflabeling. In one embodiment, a plurality of stains is used for eachround of labeling, such that each round of labeling uses a plurality ofstains. In one embodiment, at least one round of labeling includes aplurality of stains and at least one round of labeling includes a singlestain.

Amplification

In one embodiment, amplification techniques are used to increase thesignal-to-noise ratio within at least one channel or for at least onestain. The amplification techniques include, but are not limited to,addition of amplification moieties such as haptens and anti-haptens(such as DNP and anti-DNP; DIG and anti-DIG; FITC and anti-FITC; HQ andanti-HQ; and biotin), primary and secondary antibodies, horseradishperoxidase (HRP) and tyramide, HRP or alkaline phosphatase and 3,3′diaminobenzidine (DAB) or 3-amino-9-ethylcarbazole (AEC), andcomplementary molecules (such as biotin and an avidin). Theseamplification moieties bind to or interact with affinity molecules,whether directly or indirectly, that are themselves bound to or haveinteracted with one or more biomarkers.

In one embodiment, amplification is performed on a single biomarker. Inone embodiment, amplification is performed on two or more biomarkers. Inone embodiment, amplification performed on two or more biomarkers usesdifferent types of amplification techniques. In one embodiment,amplification performed on two or more biomarkers uses the same type ofamplification technique. For example, tyramide signal amplification,which includes at least one horseradish peroxidase molecule (which canbe directly or indirectly bound to or interacted with the affinitymolecule bound to or interacted with the biomarker) and at least onetyramide-detection moiety conjugate, can be used to amplify multiplebiomarkers, whether they are in the same channel or a different channel.Additionally, multiple haptens can be used to amplify multiplebiomarkers, whether they are in the same channel or a different channel.When using multiple haptens, the same type of haptens can be used toamplify different biomarkers; different types of haptens can be used toamplify different biomarkers; or two or more biomarkers may be amplifiedby a first type of hapten and one or more biomarkers may be amplified bya second type of hapten.

In one embodiment, an amplification moiety (e.g., HRP) denaturing stepcan be implemented, such as by adding hydrogen peroxide. Theamplification moiety denaturing step eliminates potentialcross-reactivity of affinity molecules while allowing for theamplification of a species-independent channel. In other words, theamplification moiety denaturing step opens up an imaging channel fordetection of an additional biomarker. In one embodiment, theamplification moiety denaturing step is performed before an antigenretrieval step, such as the second antigen retrieval step when twoantigen retrievals are performed. In one embodiment, the amplificationmoiety denaturing step is performed after an antigen retrieval step,such as the second antigen retrieval step when two antigen retrievalsare performed. The species of that affinity molecule can be independentof any other species implemented in another channel. Species, forexample, include, but are not limited to a cat, chicken, cow, dog,donkey, goat, guinea pig, hamster, horse, human, llama, monkey, mouse,pig, rabbit, rat, or sheep.

Antigen Retrieval

Antigen retrieval is a process by which epitope detectability on asample is increased. During antigen retrieval, the sample is incubatedwith a solution, such as a buffer (e.g., tris-based buffer, CellConditioning 1 from Ventana, or the like), for a given amount of time(e.g., 1 min to 24 hours, including, without limitation, 2 min, 3 min, 4min, 5 min, 10 min, 15 min, 20 min, 30 min, 45 min, 60 min, 2 hours, 3hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, and 18 hours) at agiven temperature (50-110° C., including at 50-90° C., 50-85° C., 60-80°C., 65-85° C., 70° C., 80° C., 85° C., or at 90° C.). The time andtemperature are selected based on the desired epitope or biomarker to beretrieved and appropriate conditions for renaturing the desired epitopeor biomarker. Antigen retrieval unmasks epitopes that otherwise wouldnot be detectable to a specific antibody due to sample processing, suchas fixation.

In one embodiment, antigen retrieval is implemented a single time. Inone embodiment, multiple antigen retrievals (e.g., 2, 3, 4, 5, up to 10,up to 25, up to 50, up to 100, or any desirable or appropriate amount)can be performed. In one embodiment, when multiple antigen retrievalsare performed, each successive antigen retrievals can use a highertemperature than the previous antigen retrievals. In one embodiment,when multiple antigen retrievals are performed, each successive antigenretrievals can use a lower temperature than the previous antigenretrievals. In one embodiment, when multiple antigen retrievals areperformed, successive antigen retrievals can use a higher or lowertemperature than the previous antigen retrieval based on the appropriateconditions for antigen retrieval of the desired or targetedepitope/biomarker.

In one embodiment, and as seen in FIG. 1, at least two separate antigenretrievals can be implemented to prevent a reduction or elimination ofefficacy of an antibody, detection moiety, reagent, or the like (such asby denaturing), where a particular condition (such as temperature)affects the efficacy of the antibody, detection moiety, reagent, or thelike. A first antigen retrieval is performed at approximately 50-110°C., including at 50-90° C., 50-85° C., 60-80° C., 65-85° C., 70° C., 80°C., 85° C., or at 90° C. The first antigen retrieval step is performedon or for an epitope or biomarker which can be denatured or have itsdetectability reduced when exposed to an antigen retrieval step above agiven temperature. Labeling of at least one biomarker is then performed.The labeling can include at least one amplification technique discussedabove. The labeling step, such as with the amplification step, isperformed to ensure detectability of the epitope or biomarker whenexposed to a second antigen retrieval at or above a given temperature.For example, tyramide signal amplification can be used to detect a firstbiomarker. During tyramide signal amplification, an activatedtyramide-detection moiety conjugate (by way of an enzymatic reactionwith horseradish peroxidase) forms a covalent bond with a tyrosinelocated at or near the first biomarker. The covalent bond is not brokenduring the second antigen retrieval step, thereby allowing for detectionof the first biomarker which could typically be denatured during thesecond antigen retrieval at or above the given temperature. A secondantigen retrieval is then performed at approximately 50-110° C.,including at 80-110° C. or, more specifically, at 100° C. The secondantigen retrieval is generally performed at a higher temperature thanthe first antigen retrieval. The second antigen retrieval step isperformed on or for an epitope or biomarker which is renatured (or moreoptimally renatured) at a temperature which can denature at least thefirst biomarker, as noted above. Labeling of at least one biomarker isthen performed. The labeling can include at least one amplificationtechnique discussed above. It should be noted, however, that whenantigen retrieval is performed above 100° C., a reagent can be added toincrease the boiling point of the sample, thereby reducing oreliminating any damage to the sample. The reagent can include, but isnot limited to, a glycol-based solution (e.g., propylene glycol,ethylene glycol, polyethylene glycol), glycerol or a glycerol-basedsolution, a solution including at least one ionic compound (e.g., a saltsolution), or the like. In one embodiment, no intervening orintermediary step is performed between the labeling step and the secondantigen retrieval step. In one embodiment, at least one wash step isperformed between the labeling step and the second antigen retrievalstep.

For example, a sample undergoes a first antigen retrieval process at50-90° C., 50-85° C., or 70° C. CD45, a granulocyte marker (CD11b,CD66b, or CD15), and an endothelial cell marker (CD105, CD31, or CD144)then undergo labeling. In one embodiment, the labeling incorporates anamplification technique (e.g. horseradish peroxidase and tyramide). Thesample then undergoes a second antigen retrieval process at 50-110° C.,80-100° C., or 100° C. Labeling of cytokeratin, EpCAM, a mesenchymalbiomarker, and nuclear staining is then performed, such thatcytokeratin, EpCAM, and the mesenchymal biomarker incorporated multiplehaptens, as discussed above. In this example, the granulocyte marker canbe denatured above 90° C. The first antigen retrieval step is performedat 50-90° C. Then, using tyramide signal amplification, the granulocytemarker is labeled. The CD45 and the endothelial cell marker can also belabeled with by tyramide signal amplification. The tyramide signalamplification allows for subsequent detection because the secondbiomarker, such as vimentin, requires antigen retrieval performed at atemperature above 90° C.

It may be desirable to perform antigen retrieval on a plurality ofbiomarkers, wherein each biomarker is to be retrieved or unmasked atdifferent temperatures. Starting with the lowest temperature-dependentantigen retrieval step and progressing, ultimately, to the highesttemperature-dependent antigen retrieval step. This allows for antigenretrieval of different biomarkers with at least one different antigenretrieval condition.

Some biomarkers or epitopes, for example, can be denatured by antigenretrieval processes where the temperature is greater than or equal toabout 80° C. The HRP-tyramide amplification results in activatedtyramide, via an enzymatic reaction. The activated tyramide (which isbound to a detection moiety) is deposited in the vicinity of theHRP-biomarker site and forms covalent bonds with particular proteins inthe area of deposition. The covalent bonds between the activatedtyramides and the proteins are not affected by increased temperatureswhich may occur in or are required by subsequent antigen retrievalprocesses, thereby allowing for detection even with subsequentlyelevated temperatures.

Selective Labeling

In one embodiment, a target or non-target analyte can have a biomarkerwhich is both intracellular and extracellular. In one embodiment, theratio of one biomarker to another biomarker can be of clinical utility.In one embodiment, it may be desirable to only label or preferentiallylabel one of the intracellular epitope of the biomarker or theextracellular epitope of the biomarker.

In one embodiment, such as where the target analyte (and/or non-targetanalyte, where appropriate or desirous) has been permeabilized and hasboth an intracellular epitope and an extracellular epitope, it can bedesirous to only label the extracellular epitope of the biomarker. Anantigen retrieval step is performed at 70-110° C., and typically at70-90° C. Labeling is then performed using at least one stain includingan affinity molecule directed to the biomarker linked via a linker to adetection moiety. In one embodiment, the detection can be a quantum dot.In one embodiment, the quantum dot has a diameter that is greater thanor equal to 2 nanometers. In one embodiment, the quantum dot has a shellthat is hydrophilic (e.g., polyethylene glycol (PEG)) or hydrophobic(e.g., a linker containing an alkane chain of at least four carbonatoms). In one embodiment, the quantum dot does not have a shell. In oneembodiment, the detection moiety can be an organic fluorophore. In oneembodiment, the organic fluorophore can have a molecule weight of atleast at least 1 kD, including, without limitation, at least 10 kD, atleast 25 kD, at least 50 kD, at least 75 kD, at least 100 kD, at least150 kD, at least 200 kD, at least 250 kD, at least 300 kD, at least 340kD, at least 350 kD, at least 500 kD, and at least 750 kD. The linkercan be hydrophilic (e.g., polyethylene glycol (PEG)) or hydrophobic(e.g., a linker containing an alkane chain of at least four carbonatoms). For example, the affinity molecule, the hydrophilicity of thelinker, and the size of the quantum dot directs the stain to theextracellular epitope of the biomarker and inhibits permeation of thestain across the plasma membrane. Therefore, despite permeabilization ofthe target analyte, the stain preferentially labels the extracellularepitope of the biomarker, rather than the intracellular epitope. As amore specific example, a stain including a vimentin antibody is linkedto QD800 with a PEG linker is added to a sample. A cell, having beenpermeabilized, can include an intracellular epitope of vimentin and anextracellular epitope of vimentin. However, the characteristics of thestain, as noted above (i.e., hydrophilicity of the linker and size ofthe quantum dot) are selected to induce preferential labeling of theextracellular epitope.

It should be noted that, despite the method and stain used, some of thestain can label the different epitope of the biomarker than the onedesired. However, the amount is negligible.

Algorithm

In one embodiment, after imaging, the detector transmits the data backto the computer or is retained within the scanner. The image data maythen be compiled into images, processed and analyzed by a computer orassociated software or programs, whereby the target analyte may becharacterized, such as by biomarkers present, morphology, clustering,and/or the like. The images formed due to the emission lights may beoverlaid when a plurality of fluorescent probes, having bound themselvesto the target analyte, are excited and emit light. The computer orscanner may then implement a non-transitory computer-readable mediumprocess to analyze and process the data. The non-transitorycomputer-readable medium process may detect a candidate object. In oneembodiment, target analytes are found using at least one imageprocessing technique, including, but not limited to, thresholding(manual or automatic), template matching, watershedding,cross-correlation, convolutional neural networks, region growing, andedge detection.

Non-Target Analytes

In one embodiment, non-target analytes within the sample can be labeledand detected for removal (i.e. negative marker, dumping, or dumpchannel) from analysis of the remainder of the sample, such as by imageprocessing and/or algorithm-based methods (i.e. a non-transitorycomputer-readable medium process). This increases detection of thetarget analytes by removing the undesired (or non-target) analytes fromdetermination or analysis. For example, stains including affinitymolecules for white blood cell biomarkers (such as CD45, a granulocytemarker (CD11b, CD66b, or CD15), and an endothelial cell marker (CD105,CD31, or CD144)) can be used to label and detect the white blood cellsso as to be removed from subsequent analysis.

In one embodiment, a single affinity molecule (e.g. antibody) directedto a single biomarker of a non-target analyte can be added to thesample. The single affinity molecule can be directly or indirectlylabeled with a detection moiety. An amplification technique can also beimplemented.

In one embodiment, a plurality of affinity molecules directed to aplurality of biomarkers of a non-target analyte can be added to thesample. The plurality of affinity molecules can be directly orindirectly labeled with a detection moiety. In one embodiment, eachaffinity molecule of the plurality of affinity molecules is labeled witha different detection moiety. In one embodiment, at least two affinitymolecules of the plurality of affinity molecules are labeled witheither: (1) detection moieties providing a signal in the same channel(e.g., a first affinity molecule is bound to or interacted with Alexa488 and a second affinity molecule is bound to or interacted withQD500), or (2) the same detection moiety (e.g., the affinity moleculesare bound to or interacted with Alexa 488). At least one amplificationtechnique can also be implemented for one or more of the biomarkers.

In one embodiment, the non-target analyte can express the mesenchymalcell marker.

Biological Application

FIG. 2 shows example images for objects, such that the term “object” or“candidate object” are terms (which are interchangeable) used todescribe a target analyte or non-target analyte before performinganalysis and/or scoring used to determine whether the object is a targetanalyte or non-target analyte. FIG. 2 shows labeled biomarkers (columns)of the various objects (rows).

In one embodiment, a process, as discussed below, incorporates aplurality of sub-steps (i.e. multiplexing, amplification, cycliclabeling, antigen retrieval, non-target analyte dumping, etc.) into thegeneral method. The general method can incorporate any number ofsub-steps described herein. The incorporation of these sub-steps allowsfor the detection of a target analyte having a given definition (forexample, a circulating tumor cell that is (EPCAM+ orCK+)/CD45−/nuclear+) and/or a target analyte which does not fall withinthe given definition (for example, (EPCAM or CK)+/mesenchymal+ or (EPCAMor CK)−/mesenchymal+). In one embodiment, confirmation of these targetanalytes is done by sequencing.

1. Fix sample

-   -   a. 1 hour in 10% neutral buffered formalin

2. Neutralize with buffer

-   -   b. Tris buffered saline

3. Load onto autostainer or perform manually

4. (Optional) First pre-treatment , such as to eliminate any peroxidaseor peroxidase-like compounds on or within the sample

-   -   a. Hydrogen peroxide solution        -   i. Includes sodium stannate and Target Retrieval Solution            (Dako #51699)

5. First antigen retrieval

-   -   a. Performed at 50-110° C.        -   i. Performed at 50-85° C.        -   ii. Performed at 70° C.    -   b. Include antigen retrieval solution        -   i. Such as Ventana CC1

6. Add at least one affinity molecule

-   -   a. Primary antibody        -   i. CD45        -   ii. CD11b, CD66b, or CD15        -   iii. CD105, CD31, or CD144

7. Perform amplification for the at least one biomarker

-   -   a. Add (secondary) antibody-horseradish peroxidase conjugate to        bind to or interact with the at least one antibody added in Step        6    -   b. Add tyramide-detection moiety conjugate to interact with the        (secondary) antibody-horseradish peroxidase conjugate added in        Step 7(a)

8. (Optional) Second pre-treatment, such as to eliminate any peroxidaseor peroxidase-like compounds on or within the sample

-   -   a. Hydrogen peroxide solution        -   i. Includes sodium stannate and Target Retrieval Solution            (Dako #51699)

9. (Optional) At least one wash

10. Second antigen retrieval

-   -   a. Performed at 50-110° C.        -   i. Performed at 80-110° C.        -   ii. Performed at 100° C.    -   b. Include antigen retrieval solution        -   i. Such as Ventana CC1

11. (Optional) Add another affinity molecule

-   -   a. Primary antibody        -   i. At least one of            -   1. EGFR; HER2; PSMA; Ki-67; ER; PSA

12. (Optional) Perform amplification for the at least one biomarker

-   -   a. Add (secondary) antibody-horseradish peroxidase conjugate to        bind to or interact with the at least one antibody added in Step        10    -   b. Add tyramide-detection moiety conjugate to interact with the        (secondary) antibody-horseradish peroxidase conjugate added in        Step 11(a)

13. Add at least one more affinity molecule

-   -   a. First positive marker-first hapten        -   i. CK-DIG    -   b. Second positive marker-first hapten        -   ii. EpCAM-DIG    -   c. Mesenchymal marker-second hapten        -   i. Fibronectin-biotin        -   ii. Vimentin-biotin        -   iii. N-cadherin-biotin    -   d. Nuclear stain        -   i. Sytox

14. Add first anti-hapten-detection moiety conjugate

-   -   a. Anti-DIG-Alexa 647

15. Add second anti-hapten or second hapten complementary molecule

-   -   a. Avidin-QD800        -   i. Streptavidin-QD800

16. Image or detect at least one detection moiety

When a feature or element is herein referred to as being “on” anotherfeature or element, it can be directly on the other feature or elementor intervening features and/or elements may also be present. Incontrast, when a feature or element is referred to as being “directlyon” another feature or element, there are no intervening features orelements present. It will also be understood that, when a feature orelement is referred to as being “connected”, “attached” or “coupled” toanother feature or element, it can be directly connected, attached orcoupled to the other feature or element or intervening features orelements may be present. In contrast, when a feature or element isreferred to as being “directly connected”, “directly attached” or“directly coupled” to another feature or element, there are nointervening features or elements present. Although described or shownwith respect to one embodiment, the features and elements so describedor shown can apply to other embodiments. It will also be appreciated bythose of skill in the art that references to a structure or feature thatis disposed “adjacent” another feature may have portions that overlap orunderlie the adjacent feature.

Terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention.For example, as used herein, the singular forms “a”, “an” and “the” areintended to include the plural forms as well, unless the context clearlyindicates otherwise. It will be further understood that the terms“comprises” and/or “comprising,” when used in this specification,specify the presence of stated features, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, steps, operations, elements, components, and/orgroups thereof. As used herein, the term “and/or” includes any and allcombinations of one or more of the associated listed items and may beabbreviated as “/”.

Spatially relative terms, such as “under”, “below”, “lower”, “over”,“upper” and the like, may be used herein for ease of description todescribe one element or feature's relationship to another element(s) orfeature(s) as illustrated in the figures. It will be understood that thespatially relative terms are intended to encompass differentorientations of the device in use or operation in addition to theorientation depicted in the figures. For example, if a device in thefigures is inverted, elements described as “under” or “beneath” otherelements or features would then be oriented “over” the other elements orfeatures. Thus, the exemplary term “under” can encompass both anorientation of over and under. The device may be otherwise oriented(rotated 90 degrees or at other orientations) and the spatially relativedescriptors used herein interpreted accordingly. Similarly, the terms“upwardly”, “downwardly”, “vertical”, “horizontal” and the like are usedherein for the purpose of explanation only unless specifically indicatedotherwise.

Although the terms “first” and “second” may be used herein to describevarious features/elements (including steps), these features/elementsshould not be limited by these terms, unless the context indicatesotherwise. These terms may be used to distinguish one feature/elementfrom another feature/element. Thus, a first feature/element discussedbelow could be termed a second feature/element, and similarly, a secondfeature/element discussed below could be termed a first feature/elementwithout departing from the teachings of the present invention.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising” means various components can be co-jointlyemployed in the methods and articles (e.g., compositions and apparatusesincluding device and methods). For example, the term “comprising” willbe understood to imply the inclusion of any stated elements or steps butnot the exclusion of any other elements or steps.

As used herein in the specification and claims, including as used in theexamples and unless otherwise expressly specified, all numbers may beread as if prefaced by the word “about” or “approximately,” even if theterm does not expressly appear. The phrase “about” or “approximately”may be used when describing magnitude and/or position to indicate thatthe value and/or position described is within a reasonable expectedrange of values and/or positions. For example, a numeric value may havea value that is +/−0.1% of the stated value (or range of values), +/−1%of the stated value (or range of values), +/−2% of the stated value (orrange of values), +/−5% of the stated value (or range of values), +/−10%of the stated value (or range of values), etc. Any numerical valuesgiven herein should also be understood to include about or approximatelythat value, unless the context indicates otherwise. For example, if thevalue “10” is disclosed, then “about 10” is also disclosed. Anynumerical range recited herein is intended to include all sub-rangessubsumed therein. It is also understood that when a value is disclosedthat “less than or equal to” the value, “greater than or equal to thevalue” and possible ranges between values are also disclosed, asappropriately understood by the skilled artisan. For example, if thevalue “X” is disclosed the “less than or equal to X” as well as “greaterthan or equal to X” (e.g., where X is a numerical value) is alsodisclosed. It is also understood that the throughout the application,data is provided in a number of different formats, and that this data,represents endpoints and starting points, and ranges for any combinationof the data points. For example, if a particular data point “10” and aparticular data point “15” are disclosed, it is understood that greaterthan, greater than or equal to, less than, less than or equal to, andequal to 10 and 15 are considered disclosed as well as between 10 and15. It is also understood that each unit between two particular unitsare also disclosed. For example, if 10 and 15 are disclosed, then 11,12, 13, and 14 are also disclosed.

Although various illustrative embodiments are described above, any of anumber of changes may be made to various embodiments without departingfrom the scope of the invention as described by the claims. For example,the order in which various described method steps are performed mayoften be changed in alternative embodiments, and in other alternativeembodiments one or more method steps may be skipped altogether. Optionalfeatures of various device and system embodiments may be included insome embodiments and not in others. Therefore, the foregoing descriptionis provided primarily for exemplary purposes and should not beinterpreted to limit the scope of the invention as it is set forth inthe claims.

The examples and illustrations included herein show, by way ofillustration and not of limitation, specific embodiments in which thesubject matter may be practiced. As mentioned, other embodiments may beutilized and derived there from, such that structural and logicalsubstitutions and changes may be made without departing from the scopeof this disclosure. Such embodiments of the inventive subject matter maybe referred to herein individually or collectively by the term“invention” merely for convenience and without intending to voluntarilylimit the scope of this application to any single invention or inventiveconcept, if more than one is, in fact, disclosed. Thus, althoughspecific embodiments have been illustrated and described herein, anyarrangement calculated to achieve the same purpose may be substitutedfor the specific embodiments shown. This disclosure is intended to coverany and all adaptations or variations of various embodiments.Combinations of the above embodiments, and other embodiments notspecifically described herein, will be apparent to those of skill in theart upon reviewing the above description.

The foregoing description, for purposes of explanation, used specificnomenclature to provide a thorough understanding of the disclosure.However, it will be apparent to one skilled in the art that the specificdetails are not required in order to practice the systems and methodsdescribed herein. The foregoing descriptions of specific embodiments arepresented by way of examples for purposes of illustration anddescription. They are not intended to be exhaustive of or to limit thisdisclosure to the precise forms described. Many modifications andvariations are possible in view of the above teachings. The embodimentsare shown and described in order to best explain the principles of thisdisclosure and practical applications, to thereby enable others skilledin the art to best utilize this disclosure and various embodiments withvarious modifications as are suited to the particular use contemplated.It is intended that the scope of this disclosure be defined by thefollowing claims and their equivalents:

What is claimed is:
 1. A method of detecting one or more biomarkers in asample or fraction thereof, the method comprising: providing the sampleor fraction thereof; performing a first antigen retrieval on the sampleor fraction thereof at 50-90° C. to unmask a desired epitop orbiomarker; labeling at least one biomarker of the sample or fractionthereof; performing a second antigen retrieval on the sample or fractionthereof at a temperature greater than the temperature at which the firstantigen retrieval is performed; and detecting an object expressing theat least one biomarker.
 2. The method of claim 1, wherein the firstantigen retrieval is performed at 50-85° C.
 3. The method of claim 1,wherein the at least one biomarker is CD11b, CD15, CD45, CD66b, CD105,or CD144.
 4. The method of claim 1, wherein the labeling step isperformed on a plurality of biomarkers of the sample or fractionthereof.
 5. The method of claim 4, wherein the plurality of biomarkerscomprises a white blood cell marker, a granulocyte marker, and anendothelial cell marker.
 6. The method of claim 4, wherein the detectionmoieties of the plurality of stains are in the same channel or have thesame emission wavelength.
 7. The method of claim 1, wherein the firstantigen retrieval is performed at 50-80° C.
 8. The method of claim 10,wherein the second antigen retrieval is performed at 80-110° C.
 9. Themethod of claim 10, wherein the second antigen retrieval is performed at90-100° C.
 10. The method of claim 1, wherein the labeling step furthercomprises an amplifying step.
 11. The method of claim 1, wherein thelabeling step causes a covalent bond to be formed between the sample orfraction thereof and a detection moiety.
 12. The method of claim 1,further comprising: labeling at least one other biomarker with at leastone stain, wherein the labeling of the at least one other biomarker isperformed after the second antigen retrieval.
 13. The method of claim12, the detecting step further comprising detecting the at least oneother stain.
 14. The method of claim 1, further comprising: labeling asecond plurality of biomarkers of the sample or fraction thereof,wherein labeling the second plurality is performed after the secondantigen retrieval.
 15. A method, comprising: providing a sample or afraction thereof; adding, to the sample or fraction thereof, a firstplurality of stains directed to a white blood cell marker, a granulocytemarker, and an endothelial cell marker; adding, to the sample orfraction thereof, a stain directed to a mesenchymal cell marker afterhaving performed at least one antigen retrieval; and detecting at leastone target analyte, wherein the at least one target analyte expressesexpressing the mesenchymal cell marker, and wherein the at least onetarget analyte does not express any one of the white blood cell marker,the granulocyte marker, and the endothelial cell marker.
 16. The methodof claim 15, wherein the detection moiety of the mesenchymal cell markerstain is in a different channel than the detection moieties of the firstplurality of stains.
 17. The method of claim 16, wherein the detectingstep also detects at least one non-target analyte expressing at leastone biomarker to which at least one stain of the first plurality ofstains is directed.
 18. The method of claim 15, wherein the detectionmoieties of the first plurality of stains emit wavelengths in the samechannel.
 19. The method of claim 15, wherein the first adding stepfurther comprises amplifying a signal generated by at least one of thestains.
 20. The method of claim 15, further comprising performing afirst antigen retrieval on the sample or fraction thereof at 50-90° C.,wherein the first antigen retrieval step is performed before the firstadding step.
 21. The method of claim 15, further comprising performing asecond antigen retrieval on the sample or fraction thereof at atemperature greater than the temperature at which the first antigenretrieval step is performed, wherein the second antigen retrieval stepis performed before the second adding step.
 22. The method of claim 15,wherein the target analyte is a mesenchymal cell.
 23. The method ofclaim 22, wherein the non-target analyte is a white blood cell or anendothelial cell.
 24. The method of claim 15, wherein the granulocytemarker is CD11b, CD66b, or CD15.
 25. The method of claim 24, wherein theendothelial cell marker is CD31, CD105, or CD144.
 26. The method ofclaim 15, wherein the mesenchymal cell marker is vimentin, fibronectin,or N-cadherin.
 27. The method of claim 15, wherein the second addingstep further comprises amplifying a signal generated by at least one ofthe stains.
 28. The method of claim 15, wherein the detecting stepfurther detects at least one non-target analyte expressing: at least oneof the white blood cell marker, the granulocyte marker, or theendothelial cell marker, and the mesenchymal cell marker.